A novel apparatus, the High Aspect Rotating-Wall Vessel (HARV), developed by NASA at the Johnson Space Center, Houston, TX, produces three dimensional cellular aggregates (spheroids) under low fluid shear conditIons, an aspect of simulated microgravity. The vessel rotates cells and culture medium in a disk-shaped chamber about a horizontal axis with zero headspace. Adequate gas exchange is maintained across a membrane lining one face of the chamber. Under a collaboration with NASA, CytoDiagnostics, Inc. (CDI) has studied the growth of human tumor cell lines (Glioma and Prostate) as three dimensional spheroids in the HARV apparatus. Using molecular biomarkers, CDI and its collaborators found DNA content, F-actin organization, and expression of unique tumor biomarkers correlated to in vivo cancer molecular changes, but not monolayer cultures from stationary grown tissue culture flasks. This proposal is designed to confirm and extend our observations in a bladder cell line tumor model wing proprietary tumor markers; mucins, F/G-actins organization, oncogenes, tumor suppressor genes, and epidermal growth factor and receptor probes to assess the comparisons of established known cell lines grown as two dimensional monolayers grown on Matrigel(TM) cell growth substrate versus 3-D liquid grown spheroids produced using the HARV technology. The cell lines to be employed include T-24, J-82, and several unique cell lines developed by our collaborator, Dr. Reznikoff at the University of Wisconsin, which have been extensively characterized cytogenetically. The goal is to study in vitro grown bladder cells temporally for differential, quantitatiion of in situ expressed genetic and phenotypic biomarkers. A goal will be to establish biomarker profile fidelity to known in vivo bladder tumor results established in the literature. The model will ultimately be applied to growth and expansion of small tumor biopsies for custom evaluation of new therapeutic agents. The 3D spheroid technology being developed will be provide a useful in vitro model to assess new treatments and study mechanisms of action of new and novel treatments for cancer.